New High-performance Liquid Chromatography-DAD Method for Analytical Determination of Arbutin and Hydroquinone in Rat Plasma.

Natural substances present in herbal preparations should be carefully used because they can give toxic or therapeutic effects despite of their amount or the way of administration. The safety of products of vegetable origin must be assessed before commercialisation by monitoring the active ingredients and their metabolites. This study was therefore designed to identify and quantify arbutin and its metabolite hydroquinone, naturally present in Arctostaphylos uva-ursi (L.) Spreng plant in rat plasma, after an acute and subacute administration of aqueous arbutin solution in Wistar rats. For this purpose a reversed-phase high-performance liquid chromatography coupled with photodiode array detection was developed to assess the pharmacokinetic of arbutin and hydroquinone in plasma of female rats treated with aqueous arbutin solutions. The detection (arbutin: 0.0617 µg/ml and hydroquinone 0.0120 µg/ml) and quantification (arbutin: 0.2060 µg/ml and hydroquinone: 0.0400 µg/ml) limits were determined. At the arbutin concentration level of 10.7 µg/ml repeatability was 13.33% and its recovery 93.4±6.93%, while at the hydroquinone concentration level of 10.6 µg/ml repeatability was 11.66% and its recovery 92.9±7.75%. Furthermore the method was fully validated and the obtained data indicate that the new method provides good performances.

Different dose effect of HLA-DQ alpha beta heterodimers in insulin-dependent diabetes mellitus and celiac disease susceptibility.

To compare the quantitative effect of the DQ alpha beta heterodimers DQ alpha 52 Arg+, beta 57 Asp- and DQ alpha 1*0501, beta 1*0201 on susceptibility to IDDM and CD, we characterized, at the genomic level, the DQ alpha 52 and DQ beta 57 residues of 50 IDDM Italian patients observed in Rome. The results were compared with those of a previous study concerning the oligotyping of DQ dimers in a group of CD children belonging to the same population. Our data confirm that both diseases are primarily associated with HLA-DQ alpha beta heterodimers, but the distributions of the respective susceptible DQA1 and DQB1 alleles in the two diseases were different. In fact, the highest risk of IDDM is for subjects alpha SS, beta SS that could express, by either cis- or trans-association, four susceptible heterodimers and decreases in proportion to the number of these; in regard to CD, the highest risk was found for individuals who carried only one predisposing heterodimer.

Involvement of oxygen radicals in cytarabine-induced apoptosis in human polymorphonuclear cells.

We investigated apoptosis in polymorphonuclear neutrophils (PMNs) induced by cytarabine (Ara-C). This drug increased apoptosis by 100% with respect to the controls after 3 hr of incubation. This increase was inhibited by N-acetyl-L-cysteine (NAC) or diphenyleneiodonium chloride (DPI). Ara-C alone caused an early increase (after a 30-min incubation) in intracellular oxidant generation (inhibitable by rotenone, fumonisin b1, and DPI) and in protein tyrosine phosphorylations (inhibitable by NAC). The drug also affected the observed reduction of dimethylthiazol diphenyltetrazolium bromide (MTT). No extracellular release of reactive oxygen species (ROS) was elicited by the addition of Ara-C, while the drug increased the release of ROS by N-formyl-leucyl-phenylalanine-(f-MLP) but not phorbol 12-myristate 13-acetate-stimulated PMNs. This phenomenon was abolished by the addition of genistein, whereas such an effect was not observed following the addition of 1-(5-isoquinolynilsulfonyl)-2-methylpiperazine (H7). Ara-C induced ROS release from PMNs in the presence of subthreshold concentrations of f-MLP (priming effect). These results indicate that intracellular ROS production from mitochondria promotes Ara-C-induced apoptosis. Ara-C primes plasma membranes by a mechanism involving protein tyrosine phosphorylations and may also contribute to ROS generation from the granules.

T-cell mediated autoimmunity to the insulinoma-associated protein 2 islet tyrosine phosphatase in type 1 diabetes mellitus.

The target molecules of the T-cell response in type 1 diabetes, despite their pathogenic importance, remain largely uncharacterized, especially in humans. Interestingly, molecules such as insulin and glutamic acid decarboxylase (GAD) have been shown to be a target not only of autoantibodies, but also of autoreactive T-lymphocytes both in man and in the non-obese diabetic (NOD) mouse. In the present study we aimed to determine the existence of a specific T-cell response towards the insulinoma-associated protein 2 (IA-2) islet tyrosine phosphatase, a recently identified autoantigen which is the target of autoantibodies strongly associated with diabetes development. Human recombinant IA-2 produced in Escherichia coli, was tested for its reactivity with peripheral blood lymphocytes obtained from 16 newly diagnosed type 1 diabetic patients and from 25 normal controls, 15 of whom were HLA-DR-matched. A T-cell proliferation assay was performed in triplicate employing freshly isolated cells in the absence or in the presence of the antigen to be tested (at two different concentrations: 2 microg/ml and 10 microg/ml). A specific T-cell proliferation (defined as a stimulation index (S.I.) >/=3) was observed against IA-2 used at a concentration of 10 microg/ml (but not of 2 microg/ml) in 8/16 diabetic patients, in 1/15 HLA-DR-matched control subjects (P<0.01 by Fisher exact test) and in 0/10 of the remaining normal individuals. A statistically significant difference (P<0.003 by Mann-Whitney U test) was also observed in S.I. values between patients (3.1+/-1.4) and HLA-DR-matched controls (1.7+/-0.54) employing IA-2 at a concentration of 10 microg/ml. However, when IA-2 was used at a concentration of 2 microg/ml, the difference in S. I. between patients (1.65+/-0.8) and controls (1.0+/-0.3) did not reach statistical significance. In conclusion, these data show the presence of a specific, dose-dependent T-lymphocyte response against the IA-2 islet tyrosine phosphatase at the onset of type 1 diabetes. Consequently, this molecule appears to be a target not only at the B-lymphocyte but also at the T-lymphocyte level, reinforcing the potential pathogenic role of this autoantigen in the islet destructive process.

Vitamin E and nicotinamide have similar effects in maintaining residual beta cell function in recent onset insulin-dependent diabetes (the IMDIAB IV study)

OBJECTIVE: Protection of residual beta cell function at the time of diagnosis of insulin-dependent diabetes mellitus (IDDM) by intensive insulin therapy and the addition of nicotinamide (NA) has been established. The objective of this study was to evaluate the effect of a free oxygen radical scavenger such as vitamin E (Vit E) on residual beta cell function and parameters of metabolic control in patients with recent onset IDDM undergoing intensive insulin therapy. DESIGN: The effect of Vit E was compared with that of NA (control group) in a randomized multicentre trial. METHODS: Eighty-four IDDM patients between 5 and 35 years of age (mean age 15.8 +/- 8.4 (s.d.) years) entered a one year prospective study. One group of patients (n = 42) was treated with Vit E (15 mg/kg body weight/day) for one year; the other group (n = 42) received NA for one year (25 mg/kg body weight/day). All patients were under intensive insulin therapy with three to four injections a day. Basal and stimulated (1 mg i.v. glucagon) C-peptide secretion, glycosylated haemoglobin and insulin dose were evaluated at diagnosis and at three-monthly intervals up to one year. RESULTS: Preservation and slight increase of C-peptide levels at one year compared with diagnosis were obtained in the two treated patient groups. No statistically significant differences were observed in basal or stimulated C-peptide levels between the two groups of patients for up to one year after diagnosis. Glycosylated haemoglobin and insulin dose were also similar between the two groups; however patients receiving Vit E under the age of 15 years required significantly more insulin than NA-treated patients one year after diagnosis (P < 0.04). CONCLUSIONS: Our data indicate that Vit E and NA possess similar effects in protecting residual beta cell function in patients with recent onset IDDM. Since their putative mechanism of protection on beta cell cytotoxicity is different, combination of these two vitamins may be envisaged for future trials of intervention at IDDM onset.

Anti-ganglioside antibodies in new onset type 1 diabetic patients and high risk subjects.

Insulin dependent (type 1) diabetes mellitus appears to be a genetically determined autoimmune disease. Gangliosides have been implicated in type 1 diabetes as antigenic determinants recognized by islet cell antibodies (ICA) and shown to be able to modulate autoimmune phenomena in experimental diabetes. In order to explore in type 1 diabetes the humoral immune reactivity against gangliosides, taking into account their pancreatic localization and molecular characteristics, antibodies to gangliosides GM3, GM2, GM1, GD3, GD1a, GD1b, and GT1b have been investigated in sera from new onset type 1 diabetics and relatives of type 1 diabetic patients with or without insulin (CIAA) and/or islet cell autoantibodies. Using a purposefully designed sensitive ELISA method we found that presence of antibodies directed against the pacreatic disialo-ganglioside GD3 in a significant percentage of newly diagnosed type 1 diabetics (p < 0.001 vs normal controls) but not in CIAA and/or ICA positive relatives of type 1 diabetics. These findings confirm the involvement of gangliosides in autoimmune phenomena related to type 1 diabetes and suggest disialo-ganglioside GD3 as target of a humoral immune response associated with the onset of insulin-dependent diabetes.

In vivo measurement of immunoglobulin accumulation in the pancreas of recent onset type 1 diabetic patients.

OBJECTIVE: The possibility to quantify in vivo the severity of the inflammatory process in the pancreas of patients with recent onset insulin dependent diabetes mellitus (IDDM) could be of great relevance for follow-up studies involving immunotherapy. Scintigraphy with radiolabelled human polyclonal immunoglobulins (99mTc-HIG) is currently used for the diagnosis and follow-up of several acute and chronic inflammatory diseases. In this longitudinal study we have investigated to what extent 99mTc-HIG accumulate in the pancreas of patients with recent onset IDDM and in subjects at risk to develop IDDM. METHODS: Combined computerised tomography and gamma camera imaging were used to measure the radioactivity in the pancreatic region, as the pancreas/bone radioactivity ratio (P/B). Patients with IDDM (n = 15) were investigated at the time of diagnosis and after 1 year. Five pre-diabetic ICA+ve subjects and 8 age and sex matched normal subjects were also investigated. RESULTS: Eight out of 15 newly diagnosed IDDM patients and 2/5 ICA+ve subjects showed a significant accumulation of radiolabelled HIG in the pancreas (P/B higher than the upper 1st centile of normal subjects). One year after the diagnosis a significant accumulation of immunoglobulins was still detectable in the pancreas of IDDM patients positive who were positive at diagnosis. CONCLUSIONS: These results suggest that immunoglobulins home and bind to the pancreas of patients with recent onset IDDM and also in some ICA+ve individuals. This may reflect an increased vascular permeability of pancreatic capillaries as a consequence of the inflammatory process involving the islets. Thus, this technique may be useful for monitoring the efficacy of immune intervention at diagnosis.

[Nutritional status, obesity, and metabolic balance in pediatric patients with type I diabetes mellitus]. / Stato nutrizionale, obesità ed equilibrio metabolico in soggetti in età evolutiva affetti da diabete mellito di tipo 1.

BACKGROUND AND AIMS: The aim of this study was to evaluate the nutritional status of children with type 1 diabetes and to search for possible influences of changes in body composition on aspects of diabetes. METHODS: A group of 96 diabetic subjects (41 males and 55 females) were studied, aged between 3 and 19 years old. The following parameters were examined: weight, stature, 5 skin folds, 7 circumferences, bioelectric impedance, arterial pressure, cholesterolemia, triglyceridemia, insulin dose, HbA1c and duration of disease. RESULTS: Obesity and overweight were present in 34.5% of the sample, but obesity was only observed in females (25.5%). There was also a high percentage of underweight subjects (11.5% of the entire sample). The mean values of weight BMI, 5 skin folds, 4 circumferences, FM (calculated using fold measurement and BIA) and AFA were higher in females, whereas mean values of waist/hip ratio and waist/thigh ratio and FFM (in % of body weight) were higher in males. A close correlation was also found between the 4 weight classes (underweight, normal weight, overweight, obese) and the majority of marker parameters for adiposity (5 folds, 4 circumferences, BIA, FM calculated using BIA, fold measurement and AFA). Of the other parameters examined (mean duration of disease, HbA1c assay, daily insulin dose, total cholesterolemia, triglycerididemia, arterial pressure), only the daily insulin dose showed higher values in females in 3 weight classes (underweight, normal weight and obese). Following a comparison with the control population (2469 subjects), higher mean values were found in the latter compared to diabetic subjects, but only in relation to 3 skin folds (tricipital, subscapular and suprailiac) and one circumference (forearm). CONCLUSIONS: The study shows a high frequency of overweight and obesity in children with type 1 diabetes, comparable to that in the healthy population. The finding of a higher frequency of obesity in diabetic females might be explained by their advanced puberal status, given that almost all the obese diabetic females were aged between 10 and 19 years old. The study confirms the validity of a number of anthropometric measurements (BMI, folds, circumference) and BIA in the evaluation of nutritional status in terms of body composition.

Clostridium difficile isolation in leukemic children on maintenance cancer chemotherapy. A preliminary study.

Between December 1982 and November 1983, stool specimens from 15 children with acute lymphoblastic leukemia, who were on maintenance cancer chemotherapy, were examined weekly for the presence of Clostridium difficile and its toxin. Four out of 15 patients were positive for C. difficile: three patients had stool specimens that did not contain toxin, but cultures yielded growth of toxigenic C. difficile on only one occasion. The fourth patient, who had a recent history of hospitalization, particularly aggressive cancer chemotherapy, neutropenia, and antibiotic therapy, excreted both C. difficile and its toxin for at least 1 month. All children were asymptomatic at the time of positive cultures. This preliminary study reveals a low rate of C. difficile colonization in leukemic children on maintenance cancer chemotherapy.

Association between insulin dependent diabetes mellitus and coeliac disease. A study on 175 diabetes patients.

BACKGROUND: The association between diabetes mellitus and coeliac disease has been known for many years. In a random group of 175 insulin dependent diabetes mellitus patients of varying ages the following tests have been carried out: serum antigliadin antibodies (AGA) of IgA and IgG class, antireticulin antibodies (ARA) and antiendomisyum antibodies (AEA), both of IgA class. MATERIALS AND METHODS: The patients, 85 males and 90 females, had ages ranging from 1 yr to 30 yrs (102 in paediatric age--mainly between 6 and 14 years--and 73 adults). Patients with pathological values for AEA and/or ARA underwent an intestinal biopsy. RESULTS: Out of 175 patients studied, 21 had pathological values for AEA with or without pathological values for ARA and AGA, and 2 patients had only pathological values for ARA. 23 patients (21 with pathological values for AEA with or without ARA and AGA, 2 only for ARA ) underwent intestinal biopsy, all patients with pathological values for AEA had villous atrophy. The prevalence of coeliac disease among IDDM patients was 8.8% (95% CI 3.3 to 14.3) for the children and 16.4% (95% CI 7.9 to 24.9) for the adults. In patients with mucous atrophy, ARA, AGA IgA and IgG were pathological in 85%, 71% and 61% respectively. Symptoms and insulin requirements in all patients affected by coeliac disease before and after one year on a gluten free diet were also evaluated. The patients had clinical features with prevalently one or only few atypical symptoms which disappeared on a gluten free diet. Insulin requirements after one year on a gluten free diet appeared unchanged in coeliac patients. CONCLUSIONS: The need to screen all diabetic patients for coeliac disease is underlined.

A psychotherapeutic approach with elementary school teachers.

The tradional "lectures delivering" approach to classroom teachers used by mental health practitioners is investigated in the present study. An attempt is made to demonstrate the validity of preventative work in helping elementary school teachers as important agents in the promotion of more positive mental hygiene in the classroom and by adding to the lectures the variable of group therapy--"ego-sparing" techniques type. The latter approach seems to promote a teacher's own sense of security in dealing the pupils, an easier acceptance of differences in others, and, finally, it tends to stimulate the development of a teacher's own ability to deal sensibly, more conscientiously, and more realistically with daily problems.

Polar Constituents of Calceolaria ascendens1.

A new phenylpropanoid glucoside, 1'- O-beta- D-(3,4-dihydroxy-beta-phenyl)-ethyl-4'- O-caffeoyl-beta- D-apiosyl-(1'''-->3')-glucopyranoside, named calceolarioside E, was isolated from CALCEOLARIA ASCENDENS Lind., together with two other phenylpropanoid glucosides, verbascoside and forsythoside A, and cyclohexanols rengyol, isorengyol, and 4-hydroxy-4-(2'-hydroxyethyl)-cyclohexanone.

Viral infections in transfusion-dependent patients with beta-thalassemia major: the predominant role of cytomegalovirus.

For 9 months, 38 transfusion-dependent patients with beta-thalassemia, ranging in age from 3.4 to 19.1 years, were observed for serologic evidence of viral infections, by the collection of serial serum samples. Seventy-six age-matched healthy subjects, two for each patient, were followed as controls. Samples taken at the beginning, middle, and end of the study were tested against 18 viral antigens by complement fixation (CF). In addition, tests for antibodies to HIV, Epstein-Barr virus, hepatitis A virus, and markers for hepatitis B virus were performed. When changes in the antibody titer on CF tests (greater than or equal to 2-fold increase or decrease) or persistently high titers (greater than or equal to 64) were revealed, specific enzyme immunoassays (EIAs) for IgM and IgA antibodies were performed concomitant with CF tests in all sera. When symptomatic infections occurred, viral cultures and/or direct detection of antigens were carried out by immunofluorescence methods, EIA, or latex agglutination tests. Thalassemic patients and controls had similar (p greater than 0.05) overall rates of serologically confirmed viral infections (53 versus 132), but the former group had a higher (p less than 0.01) incidence of cytomegalovirus (CMV) infections (9 versus 4). CMV infections were associated in the thalassemic patients with hepatitis (2 cases), lymphadenitis (2 cases), and upper respiratory tract infection (1 case), while the remaining cases of CMV had a subclinical course. Moreover, the thalassemic patients had a lower (p less than 0.01) incidence of symptomatic infections (27 versus 110) than controls. Therefore, this study showed that both symptomatic and subclinical CMV infections may occur often in thalassemic patients, who otherwise have subclinical viral infections at an overall rate similar to that in healthy subjects.

Peritoneal dialysis in an infant with type 1 diabetes and hyperosmolar coma.

Hyperosmolar coma which is characterized by severe hyperglycemia in absence of chetosis is very rare in pediatric age with only 11 cases reported in the literature. The outcome of the condition is usually poor with mental retardation being the most common event. Here a case of hyperosmolar coma is described in a female of three months of age who was treated with peritoneal dialysis 11 hours after admittance to hospital. This female patient has been receiving insulin from three months of age and today at the age of 10 years she leads a normal life despite being on insulin therapy. A very low level of C-peptide (<0.3 ng/ml) clearly confirms she is affected by Type 1 diabetes. To our knowledge this is the first case report of hyperosmolar coma in a neonate with Type 1 diabetes who survived this condition without late neurological consequences.

[Difference between arterial and end-tidal carbon dioxide tension during surgery of lumbar herniated disk in general anesthesia]. / Differenza fra tensione di anidride carbonica arteriosa e di fine espirazione durante intervento per ernia discale lombare in anestesia generale.

OBJECTIVE: To determine the correlation of PaCO2 and ETCO2 during operations for lumbar disk herniation in prone position: the influence of position and deliberate hypotension. DESIGN: Prospective, to compare PaCO2, ETCO2, pH, SBE, Pa max, in a group of 10 patients undergoing elective intervention for lumbar disk herniation in prone position and in a control group of 10 patients undergoing interventions for elective non traumatologic orthopaedic surgery of the lower limbs in supine position. SETTING: Orthopaedic department of a non teaching hospital. MEASUREMENTS AND MAIN RESULTS: The results for the patients of the supine group were: mean PaCO2 35.26 mmHg (SD 3.045), mean P(a-ET)CO2 3.46 mmHg (SD 1.898), mean pH 7.433 (SD 0.044), mean SBE - 1.16 (SD 1.718), mean Pa max 122.5 mmHg (SD 17.989). In the prone group: mean PaCO2 30.3 mmHg (SD 5.819), mean P(a-ET)CO2 1.4 mmHg (SD 4.445), mean pH 7.430 (SD 0.052), mean SBE-3.93 (SD 3.255), mean Pa max 100.3 (SD 10.945). The difference was significant (p < 0.05) for pH, PaCO2, SBE; in the prone group the variability of P(a-ET)CO2 was greater and the values related with SBE. CONCLUSIONS: ETCO2 is a useful monitoring for PaCO2 in the situation evaluated but the accuracy of the correlation with PaCO2 is lesser than during standard surgical techniques, the metabolic acidosis observed is probably related to the effects of the peculiar position and the anaesthetic technique.

Two New Glucosides from Hypoxis obtusa: Obtuside A and Obtuside B1,5.

From the rhizomes of HYPOXIS OBTUSA two monoglucosides having the same aglycone as hypoxoside, 1-(3',4'- dihydroxyphenyl)-5-(3'',4''-dihydroxyphenyl)-1-pen-ten-4-yne, have been obtained. By methylation followed by hydrogenation they have been transformed into the same compound, 1-(3'-methoxy-4'- O-beta- D-glucopyranosyl-phenyl)-5-(3''-4''-dimethoxyphenyl)-pentane, likewise obtainable through partial hydrolysis of hypoxoside.

Diffusing capacity for carbon monoxide in children with type 1 diabetes.

AIMS/HYPOTHESIS: Few data are available on lung dysfunction in children with diabetes. We studied the association of pulmonary function variables (flows, volumes and alveolar capillary diffusion) with disease-related variables in children with type 1 diabetes mellitus. METHODS: We studied 39 children with type 1 diabetes (mean age 10.9+/-2.6 years, disease duration 3.6+/-2.4 years, insulin.kg(-1).day(-1) 0.77+/-0.31) and 30 healthy control children (mean age 10.4+/-3.0 years). Pulmonary function tests included spirometry, N(2) wash-out and the single-breath diffusing capacity for carbon monoxide (DL(CO)) corrected for the alveolar volume (DL(CO)/V(A)). Glycaemic control was assessed on the basis of HbA(1)c, with HbA(1)c values of 8% or less considered to indicate good glycaemic control, and HbA(1)c values of 8% or more considered to indicate poor control. RESULTS: Children with poor glycaemic control had comparable percentage values for predicted flows and volumes but lower DL(CO)/V(A) values than children with good glycaemic control and healthy control children (86.7+/-12.6 vs 99.8+/-18.4 and 102.0+/-15.7; p<0.05). The predicted DL(CO)/V(A) percentages correlated with HbA(1)c levels (r=-0.39, p=0.013). A multiple regression analysis (stepwise model) controlling for HbA(1)c levels and other disease-related variables (age of disease onset, disease duration, daily insulin dose/kg, sex) identified HbA(1)c levels as the sole predictor of DL(CO)/V(A) in percent. CONCLUSIONS/INTERPRETATION: In children with type 1 diabetes, the diffusing capacity diminishes early in childhood and is associated with poor metabolic control. Although low DL(CO)/V(A) levels in these children probably reflect pulmonary microangiopathy induced by type 1 diabetes, other factors presumably influencing CO diffusion capacity measurements (e.g. a left shift in HbA(1)c resulting in high O(2) binding and low CO binding) could explain the apparent capillary and alveolar basal membrane dysfunction.